Jul 11, 2014
Top-down meets bottom-up to make arrays of single-molecule detection wells
Single-molecule imaging has provided an important tool for a range of biological studies and technology developments, such as real-time DNA sequencing. However, the challenge in a biological system is not always detecting the molecules, but making sure just a single molecule, and not the ones around it, is detected. Now, researchers in Germany have combined top-down with bottom-up approaches to position and isolate individual fluorophore molecules in an array of wells.
"This procedure can improve the efficiency of DNA sequencing and also be beneficial for applications in other areas of research like molecular electronics," says Philip Tinnefeld of Braunschweig University of Technology.
Single-molecule imaging is used in real-time DNA sequencing to determine the precise order of the nucleotides, the building blocks of DNA. By observing a single polymerase, the molecule responsible for duplicating a strand of DNA, it is possible to identify the nucleotides that have just been added through the copying process.
"Monitoring the incorporation of single nucleotides into a full DNA strand in real-time is a revolutionary method," notes Tinnefeld. "It's almost a live broadcast."
But the technique relies on detecting a single polymerase at a time: if a second polymerase is present, the two signals are mixed up and DNA sequence information is lost. With the new work Tinnefeld and his team have made it easier to isolate and detect single molecules by developing a way to control their positioning with nanoscale precision.
The conventional approach used by researchers to capture signals from a single polymerase has been to exploit glass slides covered with a thin metal coating patterned with nanometre-sized holes. At low concentrations, some of the wells, just over a third at best, contain a single polymerase while the rest are empty or contain more than one molecule.
As the slide is illuminated, light propagates into the wells but is prevented from escaping because the well diameter is smaller than the wavelength of light. This allows the molecules to be detected. "The challenge for this application is to equip each of these nano-holes with exactly one polymerase," explains Tinnefeld.
Tinnefeld's team have updated this technique with "DNA origami" – self-assembling collections of synthetic DNA that can form shapes around 100 nm in size, roughly the size of the wells. Tinnefeld’s lab formed tiles from DNA origami with a single fluorescent molecule attached to them. The origami tiles were designed to stick to the bottom of a well, taking up the entire area of the well and preventing subsequent origami from binding. This ensures that there is a single fluorescent molecule in each well.
Matching the size of the wells to the origami size resulted in 60% of wells containing a single fluorescent molecule. When the researchers varied the size of the wells, they observed more occurrences of two origami in the larger wells and no molecules entering the smaller wells.
The results demonstrate the use of DNA origami to position single molecules in nanoscale wells. Tinnefeld points out that the technique could be extended to other single-molecule systems, such as the polymerase used for sequencing.
Full details are reported in Nano Lett. 14 3499–3503.
About the author
Richard Muscat is a postdoctoral researcher at the University of Washington.